Poor wound healing is a major health issue in insulin-resistant diabetes. Improved understanding of wound pathology, including the function of keratinocytes (KCs) in re-epithelialization, and new interventions for impaired wound healing are needed. Ganglioside GM3 is a glycosphingolipid that regulates receptor signaling at the membrane level. We have found that GM3 and GM3 synthase (GM3S) are increased in human and mouse diabetic skin. In addition, TNF?? and excess glucose-induced insulin resistance in KCs is associated with increased GM3 and reversed by GM3 depletion, suggesting that GM3 mediates diabetic wound healing impairment. Wounds heal normally in diet-induced obese (DIO) GM3 synthase knockout (GM3S-/-) mice, in contrast to delayed healing in DIO wildtype (WT) littermates. Furthermore, we have replicated reversal of this wound healing improvement in WT DIO diabetic mice by depleting GM3 with topically applied GM3S siRNA spherical nuclei acid (SNA) nanoparticle conjugates. Depleting GM3 by either GM3S SNA or GZ 161, a glucosylceramide synthase inhibitor, accelerates migration in 2D keratinocyte cultures by activating insulin-like growth factor-1 receptor (IGF1R) and Rac1 signaling. Increases in GM3, including by treatment with excess glucose or chronic, low-dose TNF?, suppress IGF1R signaling and inhibit KC migration. Our long-term goals are to test GM3 depletion in skin as a novel means to reverse the impaired wound healing in diabetics and understand how GM3 inhibits IGF1R activation and suppresses keratinocyte migration. Our first aim is to optimize GM3 depletion-based intervention by comparing the ability of topical GM3S siRNA SNA and topical or oral GZ 161 to promote healing. We will test these therapeutic options in a DIO mouse with fluorescent sensory nerves to track the impact of treatment on cutaneous innervation. The most efficacious therapy will then be studied in a diabetic model with more chronic wounds, the biofilm-challenged db/db mouse. Using normal and diabetic 3D co-culture wound models, we will validate our observations in 2D KCs and will investigate the effects of GM3 modulation on the insulin/IGF-1 signaling axis (Aim 2). Finally, as our third aim and with a focus on IGF1R, we will elucidate the role of GM3 in regulating the membrane-based localization, dynamics, and molecular interactions of insulin/IGF-1 signaling. We will interrogate the direct interactions of IGF1R with GM3 and test our hypothesis that increased GM3 prevents IGF1R clathrin-mediated endocytosis and signaling. Finally, we will use FLIM to determine if GM3 depletion increases IR-IGF1R hybrid receptors to boost IGF-1 responses. These proposed studies will increase our understanding of the membrane-based dynamics that impact insulin/IGF-1 signaling in skin. Furthermore, acceleration of wound healing, whether by nano-delivery of gene suppression or small molecule inhibition of GM3 synthesis, could be fast-tracked towards translational application as a new treatment approach for diabetic wounds.